The objective of this study was to investigate the localization and hormonal regulation of smooth muscle myosin II (SMM II) and a smooth muscle actin (αSMA) in the baboon uterus, since cytoskeletal proteins are involved in secretory function and morphological transformation. Uterine tissue was obtained from baboons 1) during the menstrual cycle, 2) following steroid treatment of ovariectomized baboons, 3) during pregnancy (Days 14–60 postovulation [PO]), and 4) during simulated pregnancy (Days 18–32 PO). Tissues were processed for immunocytochemical localization of SMM II or αSMA with specific polyclonal or monoclonal antibodies, respectively. SMM II stained all smooth muscle cells of blood vessels and myometrium regardless of treatment. Glandular epithelial staining was present only in endometrium obtained during the luteal phase or following estrogen and progesterone treatment. Staining intensity was greater in the basalis than in the functionalis. The number of glands staining positive for SMM II on Days 18–32 of pregnancy and simulated pregnancy was variable. Glandular stain was absent after Day 32 PO. These immunocytochemical data were confirmed by immunoblot analysis of glandular cytosolic extracts. Stromal staining for SMM II was present underthe luminal epithelium during simulated pregnancy (Days 18–32), on Day 25 of steroid treatment in the simulated-pregnant controls, and in nonimplantation sites during pregnancy. In contrast, SMA staining was low or absent in all uterine cell types in ovariectomized baboons. Under estrogen-dominated conditions (follicular phase and estrogen treatment), αSMA staining was present in smooth muscle cells, and this staining persisted throughout the remaining treatment periods. Glandular epithelial staining for αSMA was absent in all treatment groups. However, αSMA staining in stromal fibroblasts underneath the luminal epithelium was evident as early as Day 14 of pregnancy and Day 18 of simulated pregnancy. The number of stromal fibroblasts that stained positive increased in the surface region of the functionalis between Days 18 and 32 PO, and the staining extended throughout the upper functionalis region. There was a decrease in the number of positively stained stromal fibroblasts, particularly at the implantation site, between Days 32 and 40 of pregnancy. By Days 50–60 of pregnancy, this staining was almost absent. The induction of αSMA in stromal fibroblasts in the functionalis region in pregnant baboons was confirmed by immunoblot analysis of stromal cell cytosol extracts. We conclude that the progesterone-induced glandular expression of SMM II may be involved in uterine secretory function and that aSMA expression in stromal fibroblasts during pregnancy and after long-term steroid treatment is associated with the decidualization process.